The incidence of cancer increases with age, suggesting that multiple events (DNA mutations) must take place for malignant transformation to occur. It is known that natural defense mechanisms serve to protect against accumulation of DNA mutations and to diminish the risk of developing cancer. Defense mechanisms are of particular importance in tissues with high cellular turnover, such as the skin, intestines, and the hematopoietic tissue, because each round of replication provides enormous opportunity to generate mutations. Currently, our understanding of how the genomic integrity in the different tissues is maintained during aging is limited largely to analyses of DNA damage, DNA repair, and mutagenesis in differentiated cells and tissues. There is increasing evidence pointing to the stem cells as important regulators of genome integrity for the tissues they reside in. We therefore hypothesize that it is a failure in stem cell function that links the increasing cancer incidence with aging. We will test this premise by measuring mutant frequency in hematopoietic stem cells and compare this to that measured in differentiated cells of the myeloid - and B-cell lineage. By obtaining purified cell populations from mice of 4 age groups, day 14.5 of gestation, young adult (8-10 weeks), middle-aged (15 months), and old (26- to 28 months), we will furthermore determine whether the mutant frequency within a particular cell population changes with age. As a read-out for mutagenesis we will use the spontaneous mutant frequency. Mice transgenic for a mutation reporter gene, LacI, will be used as bone marrow donors for the isolation of purified stem cells and differentiated populations. The transgene in the cells of these mice can be isolated and the presence of spontaneous mutants determined. It is well established that hematopoietic stem cell function is greatly affected by the microenvironment they reside in. To test whether the hematopoietic stem cell microenvironment also affects the genomic integrity of HSCs, we will furthermore follow the spontaneous mutation frequency in hematopoietic stem cells (isolated from LacI transgenic mice) that are subjected to 4 rounds of serial transplantation in wild- type mice versus mice heterozygous for the CREB binding protein (CBP). The latter was chosen because CBP mice show microenvironment-mediated loss of hematopoietic stem cell support and excess myeloid differentiation. With these experiments we will initiate a line of research that will eventually lead to a systematic analysis of cell intrinsic and extrinsic mediators of DNA damage and mutagenesis as a function of aging in HSCs. This is of importance because it may lead to the development of novel cancer-preventive measures. PUBLIC HEALTH RELEVANCE: The relative number of people above 65 years in the United States of America is steadily increasing, and thus cancer is a serious health issue. With this proposal we will obtain knowledge that will significantly contribute to our currently limited understanding of the positive correlation between cancer risk and age, possibly leading in the future to the development of more cancer-preventive measures.